Multifunctional polymeric tissue coatings

ABSTRACT

Compositions for coating biological and non-biological surfaces, which minimize or prevent cell-cell contact and tissue adhesion, and methods of preparation and use thereof, are disclosed. Embodiments include polyethylene glycol/polylysine (PEG/PLL) block or comb-type copolymers with high molecular weight PLL (greater than 1000, more preferably greater than 100,000); PEG/PLL copolymers in which the PLL is a dendrimer which is attached to one end of the PEG; and multilayer compositions including alternating layers of polycationic and polyanionic materials. The multi-layer polymeric material is formed by the ionic interactions of a polycation and a polyanion. The molecular weights of the individual materials are selected such that the PEG portion of the copolymer inhibits cellular interactions, and the PLL portion adheres well to tissues. The compositions and methods are useful, for example, in inhibiting formation of post-surgical adhesions, protecting damaged blood vessels from thrombosis and restenosis, and decreasing the extent of metastasis of attachment-dependent tumor cells. The compositions and methods are also useful for coating non-biological surfaces such as metallic surfaces.

BACKGROUND OF THE INVENTION

[0001] This application is generally in the area of biocompatiblepolymeric materials which can be applied to biological andnon-biological surfaces to minimize cell-cell interactions and adhesionof cells or tissue to the surfaces.

[0002] There is a need for materials, and methods of use thereof, whichcan be used to encapsulate cells and tissues or biologically activemolecules which are biocompatible, and which do not elicit specific ornon-specific immune responses. An important aspect of the use of thesematerials in vivo is that they must be applied within the time of ashort surgical procedure or before the material to be encapsulateddisperses, is damaged or dies.

[0003] It is often desirable to implant exogenous cells into a patient,for example, to produce various products the patient is incapable ofpreparing. An example of this is implantation of exogenous Islets ofLangerhans cells to produce insulin in a diabetic patient. However,unless protected, exogenous cells are destroyed immediately followingtransplantation. Numerous attempts have been made to encapsulate thecells to minimize the body's efforts to destroy them.

[0004] Cells have been encapsulated using the ionic crosslinking ofalginate (a polyanion) with polylysine or polyornithine (polycation)(Goosen, et al., Biotechnology and Bioengineering, 27:146 (1985)). Thistechnique offers relatively mild encapsulating conditions. Microcapsulesformed by the coacervation of alginate and poly(L-lysine) have beenshown to be immunoprotective. However, the capsules do not remain intactlong after implantation, or are quickly surrounded by fibrous tissue.

[0005] The biocompatibility of alginate-poly(L-lysine) microcapsules hasbeen reported to be significantly enhanced by incorporating a graftcopolymer of PLL and PEO on the microcapsule surface (Sawhney, et al.,Biomaterials, 13, 863-870 (1991)). The PEO chain is highly water solubleand highly flexible. PEO chains have an extremely high motility in waterand are essentially non-ionic in structure. Immobilization of PEO on asurface has been largely carried out by the synthesis of graftcopolymers having PEO side chains.

[0006] U.S. Pat. Nos. 5,573,934 and 5,626,863 to Hubbell et al. disclosehydrogel materials including a water-soluble region such as polyethyleneglycol and a biodegradable region, including various biodegradablepolymers such as polylactide and polyglycolide, terminated withphotopolymerizable groups such as acrylates. These materials can beapplied to a tissue surface and polymerized, for example, to form tissuecoatings. These materials are adhered to tissue surfaces by polymerizingthe photopolymerizable groups on the materials after they have beenapplied to the tissue surface.

[0007] U.S. Pat. No. 5,627,233 to Hubbell et al. disclosesmultifunctional polymeric materials for use in inhibiting adhesion andimmune recognition between cells and tissues. The materials include atissue binding component (polycation) and a tissue non-binding component(polynonion). In particular, Hubbell discloses various PEG/PLLcopolymers, with molecular weights greater than 300, with structureswhich include AB copolymers, ABA copolymers, and brush-type copolymers.These polymers are being commercially developed for use as tissuesealants and to prevent surgical adhesions.

[0008] It is therefore an object of the present invention to provide apolymeric material that can be applied to living cells and tissues, in avery short time period, to protect the cells and tissues from cell tocell interactions, such as adhesion.

[0009] It is a further object of the present invention to provide apolymeric material which is biocompatible and resistant to degradationfor a specific time period.

[0010] It is a further object of the present invention to providecompositions for inhibiting tissue adhesion and cell-cell contact withinthe body, as well as methods for making and using the compositions.

SUMMARY OF THE INVENTION

[0011] Compositions for encapsulating cells and for coating biologicaland non-biological surfaces, which minimize or prevent cell-cell contactand tissue adhesion, and methods of preparation and use thereof, aredisclosed. Embodiments include polyethylene glycol/polylysine (PEG/PLL)block or comb-type copolymers with high molecular weight PLL (greaterthan 1000, more preferably greater than 100,000); PEG/PLL copolymers inwhich the PLL is a dendrimer which is attached to one end of the PEG;and multilayer compositions including alternating layers of polycationicand polyanionic materials. In the PEG/PLL dendrimers, the molecularweight of the PLL is between 1,000 and 1,000,000, preferably greaterthan 100,000, more preferably, between 300,000 and 800,000, and themolecular weight of the PEG is between 500 and 2,000,000, morepreferably between 5,000 and 100,000. For PEG of MW 5000, the optimalratio is between 1 PEG chain for every 3 to 10, preferably 5 to 7,lysine subunits. The optimal ratio for PEG of a molecular weight otherthan 5000 can be determined using routine experimentation, for example,using the procedures outlined in Example 1. In general, PEG/PLL graftsof various ratios are synthesized, for example, by varying the relativestoichiometric amounts of each component used in a suitable couplingreaction, and their relative efficacy in preventing a model bindinginteraction can then be determined. One method for doing this involvesdetermine the extent of cell spreading on an anionic polystyrenesurface, either uncoated or coated with the polymers.

[0012] The dendrimer is covalently grafted to one end of a PEG block.The dendrimer is a lysine dendrimer which preferably contains between 16and 128 reactive amine groups, which correlates to a dendrimer ofbetween generation 4 and generation 7. The molecular weight of the PEGis between 500 and 2,000,000, preferably between 5,000 and 100,000.

[0013] The multi-layer polymeric material is formed by the ionicinteractions of a polycation and a polyanion. There are preferablygreater than five alternating layers, more preferably more than tenalternating layers, and most preferably, greater than fifteenalternating layers of the polycationic and polyanionic materials. In apreferred embodiment, the topmost and/or bottommost layers are preparedfrom materials which include a polycationic tissue binding domain and anonionic non-tissue binding domain, such as PEG/PLL copolymers.

[0014] The polymer is applied in a fluid phase to the tissues or cellsto be protected, whereupon the tissue binding domains adsorb thepolymeric material to the tissue. The fluid phase can be applied toisolated tissue or to tissue during surgery or by means of a catheter orother less invasive device.

[0015] The PEG/PLL copolymers can be used for inhibiting cell-cellcontact and tissue adhesion. The PLL polymer adsorbs to cells or tissue,and the PEG polymer does not adsorb to tissue. When the two-domainpolymeric material contacts a tissue surface, the tissue-bindingdomain(s) binds and immobilizes the attached non-binding domain(s),which then generally extends away from the tissue surface and stericallyblocks the attachment of other tissues.

[0016] The materials can be applied to isolated tissue or to tissueduring surgery or by means of a catheter or other less invasive device.The compositions are useful for blocking adhesion and immune recognitionand thus may be useful in the treatment of many diseases andphysiological disorders, including the prevention of postoperativeadhesions, protecting injured blood vessels from thrombosis and intimalthickening relating to restenosis, and decreasing the extent ofmetastasis of tumor cells in tissues. The materials can be used, forexample, as semipermeable membranes, as adhesives as tissue supports, asplugs, as barriers to prevent the interaction of one cell tissue withanother cell or tissue, and as carriers for bioactive species. A widevariety of biological and non-biological surfaces, with differentgeometries, can be coated with these polymeric materials.

BRIEF DESCRIPTION OF THE FIGURES

[0017] FIGS. 1A-D are graphs of the spreading of human foreskinfibroblast cells (HFF cells) (cells per mm²) on various substratescoated versus the number of polyelectrolyte bilayers. In FIG. 1A, thesubstrate is tissue culture plates formed of polystyrene which has beenrendered anionic by surface treatment (TCPS) and the electrolytes arepolylysine (PLL) and alginate. In FIG. 1B, the substrate is TCPS coatedwith gelatin and the electrolytes are PLL and alginate. In FIG. 1C, thesubstrate is human foreskin fibroblast cells extracellular matrix (HFFECM) and the electrolytes are PLL and alginate. In FIG. 1D, and theelectrolytes are polyethylene imine (PEI) and polyacrylic acid (PAA).The squares represent spread cells, and the circles represent adheredcells.

[0018]FIG. 2 is a graph of the thickness (Angstroms) of (PLL/alginate)polyelectrolyte bilayers versus the number of bilayers coated on anSi/SiO₂ substrate (circles) and Si/SiO₂ wafers coated with gelatin(squares).

[0019]FIG. 3 is a graph of the number of spread cells/mm² after a fourhour incubation versus the ratio of lysine to PEG in various graftcopolymers. Squares represent copolymers of mPEG with a molecular weightof 5000 and PLL with a molecular weight of 20,000. Circles representcopolymers of MPEG with a molecular weight of 5000 and PLL with amolecular weight of 375,000.

DETAILED DESCRIPTION OF THE INVENTION

[0020] Compositions for encapsulating cells and for coating biologicaland non-biological surfaces, which minimize or prevent cell-cell contactand tissue adhesion, and methods of preparation and use thereof, aredisclosed. The compositions are either specific PEG/PLL copolymers ormultilayer materials formed of alternating layers of polyelectrolytes.The compositions can be used, for example, to plug, seal, support orcoat tissue or other surfaces to alter cellular adhesion to the surface.

[0021] I. Compositions

[0022] The materials are biocompatible. Materials are consideredbiocompatible if the material either elicits a reduced specific humoralor cellular immune response or does not elicit a nonspecific foreignbody response that prevents the material from performing the intendedfunction, and the material is not toxic upon ingestion or implantation.The material must also not elicit a specific reaction such as thrombosisif in contact with the blood.

[0023] A. PEG/PLL Copolymers.

[0024] 1. Polymer Composition

[0025] The following definitions apply to the PEG/PLL copolymersdescribed herein. Block copolymers are defined as copolymers in which apolymeric block is linked to one or more other polymeric blocks. This isdistinguished from random copolymers, in which two or more monomericunits are linked in random order to form a copolymer. Brush copolymers(as in a bottle brush) are copolymers which have a backbone of onecomposition and bristles of another. These copolymers are also known ascomb copolymers. The terms brush and comb are used interchangeably.Dendritic polymers, also known as dendrimers or starburst polymers, arepolymers which include a core molecule which is sequentially reactedwith monomers with three or more reactive groups, such that at eachsequential coupling step, the number of reactive groups at the ends ofthe polymer increases, usually exponentially. A dendron is a subunit ofa dendrimer, the cone shaped structure resulting from sequentialreactions starting with a core containing only reactive group. As usedherein, molecular weight refers to weight average molecular weight,unless otherwise specified. As used herein, PEG is an abbreviation forpolyethylene glycol, also known as polyethylene oxide orpolyoxyethylene. The phrase “(meth)acrylic” refers to either acrylic ormethacrylic groups.

[0026] a. PEG/PLL Brush or Comb-Type Graft Copolymers.

[0027] The PEG/PLL co-polymers can be brush copolymers (as in a bottlebrush, with a backbone of one composition and bristles of another) witha backbone of polylysine (PLL) and bristles of polyethylene glycol(PEG). The molecular weight of the PLL is between 1,000 and 1,000,000,preferably greater than 100,000, more preferably, between 300,000 and800,000. The molecular weight of the PEG is between 500 and 2,000,000,more preferably between 5,000 and 100,000.

[0028] For PEG with a MW of 5000, the optimal graft ratio is between 1PEG chain for every 3 to 10, preferably 5 to 7, lysine subunits for invitro models, and may be adjusted based on desired properties for invivo applications. However, for PEG with a different molecular weight,the optimal ratio is expected to change. Optimization of the polymers isdiscussed, for example, in Examples 1 and 2.

[0029] Various tissue binding polycationic polymers can be substitutedfor PLL, and various non-tissue binding polymers can be substituted forPEG.

[0030] Suitable polycationic blocks include natural and unnaturalpolyamino acids having net positive charge at neutral pH, positivelycharged polysaccharides, and positively charged synthetic polymers.Representative polycationic blocks include monomeric units selected fromthe group consisting of lysine, histidine, arginine and ornithine.Representative positively charged polysaccharides include chitosan,partially deacetylated chitin, and amine-containing derivatives ofneutral polysaccharides. Representative positively charged syntheticpolymers include polyethyleneimine,

[0031] polyamino(meth)acrylate, polyaminostyrene, polyaminoethylene,poly(aminoethyl)ethylene, polyaminoethylstyrene, and N-alkyl derivativesthereof. Suitable non-tissue binding polymers include mixed polyalkyleneoxides having a solubility of at least one gram/liter in aqueoussolutions, neutral water-soluble polysaccharides, polyvinyl alcohol,poly-N-vinyl pyrrolidone, non-cationic poly(meth)acrylates andcombinations thereof can be substituted for PEG.

[0032] For example, PEG reacted with polyethylene imine with a molecularweight greater than 10,000 will have approximately the same physicalproperties as the PEG/PLL copolymers described herein. Polyhydroxyethylmethacrylate can be reacted with a suitable stoichiometric ratio of areagent such as tresyl or tosyl chloride (an activating agent), whichconverts some of the hydroxy groups to leaving groups. These leavinggroups can be reacted with polycationic polymers, for example,polyaminoethyl methacrylate with a molecular weight greater than 10,000,to yield a high molecular weight polymer. A suitable stoichiometricratio is one mole activating agent per mole of polyhydroxyethylmethacrylate, and one mole activated polyhydroxyethyl methacrylate perevery 3 to 9, preferably 5 to 7 moles of reactive groups onpolyaminoethyl methacrylate. Suitable cationic polymers are those that,when combined with a suitable non-tissue binding polymer, have roughlythe same physical properties as the PEG/PLL copolymers described herein.

[0033] b. PEG/PLL Dendrimers.

[0034] The PEG/PLL dendrimers are copolymers where one or more linearPEG polymeric blocks are covalently linked to the focal point of acationic dendrimer, for example, dendrimerically polymerized polylysine,such that the dendrimer fans out from the PEG. Preferably, the PEG islinked to the central point of the dendrimer, which is grown from thePEG as described in detail below. The particular utility of thedendritic construction is the ability to precisely control the mass ofthe resulting copolymer, the geometrical relationship between thepolymeric blocks, and the degree of substitution. For instance, in theexamples shown, there is exactly one PEG for a defined number ofpositive charges. In contrast, grafting preformed PEG molecules onto apolycationic backbone normally results in a random positioning of thePEG groups on the backbone.

[0035] The dendrimer preferably contains between 16 and 128 reactiveamine groups, which correlates to a dendrimer of between generation 4and generation 7. The molecular weight of the PEG is between 500 and2,000,000, preferably between 5,000 and 100,000.

[0036] The amine groups in copolymers listed in the Examples are theprimary amines of lysine residues, but other groups can be used. Forexample, the last “generation” of the polymer can be prepared usingarginine or histidine, resulting in guanidino or imidazoyl cationicgroups, respectively. Likewise, more than one PEG group can be provided,for example, by using as a starting material a small molecule with atleast two carboxyl groups and at least two amino groups, for example,the dipeptide Glu-Lys.

[0037] For all embodiments, the molecular weight and number of PEGblocks per lysine block is determined such that the resulting copolymerhas the properties of both the PLL and the PEG. If the proportion of PEGis too high, the bioadhesion of the polymer is reduced. If theproportion of PLL is too high, the ability of the PEG to minimizecell-cell interactions and tissue adhesion is reduced. The polymers musthave sufficient PEG character to minimize cell-cell and tissueinteractions. Polymers with too few PEGs per PLL are less suitable forminimizing these interactions. The polymers must also have sufficientPLL character to adequately bind to a tissue or cell surface. Polymerswith insufficient PLL character fail to bind adequately to a tissue orcell surface.

[0038] Although the copolymers are described above with respect to PEGand PLL, the same activities can be obtained from variants of thesepolymers. Various non-tissue binding polymers can be used in place of orin addition to PEG, for example, polyalkylene oxides having a solubilityof at least one gram/liter in aqueous solutions, such as some poloxamernonionic surfactants, many neutral polysaccharides, including dextran,ficoll, and derivatized celluloses, polyvinyl alcohol, non-cationicpolyacrylates, such as poly(meth)acrylic acid, and esters amide andhydroxyalkyl amides thereof, and combinations thereof. The polycationicpolymer can be any biologically acceptable polycation that provides asufficient amount and density of cationic charges to be effective atadhering to cells and tissue. A number of suitable compounds are listedbelow in section B(1).

[0039] The dendrimeric PLL allows the formation of a compact structure,with a high charge density. These PEG-lysine dendrons are effective inpreventing cell spreading when adsorbed to a simple anionic surface ifthe polymer contains about 8 or more positive charges (generation 3dendron).

[0040] The copolymer can prevent hemagglutination of human red bloodcells by a lectin, if, in the presence of PEG-lysine dendron at aconcentration in aqueous solution of 1% or greater, if the polymercontains about 32 or more positive charges (generation 5 dendron).

[0041] It has been assumed by others that PLL of MW higher than 40,000could not be used to synthesize PLL-PEG graft copolymers (PLL-g-PEG),because of the toxicity of higher MW PLL. However, PLL-g-PEG isextremely well tolerated by cells in culture, in contrast to PLL.PLL-g-PEG copolymers with a PLL backbone of MW 375,000 exhibit enhancedefficacy in some in vitro models, presumably due to enhanced adsorptionto biological surfaces as compared with PLL-g-PEG copolymers of lowerMW. PLL-g-PEG copolymers with backbones of PLL with MW 375,000 were ableto prevent fibroblast spreading onto surfaces containing pre-adsorbedserum proteins, and additionally can prevent the recognition of redblood cell surfaces by lectins. The relatively high MW PLL backbone(greater than 1,000, preferably greater than 100,000) is necessary toachieve these results.

[0042] Other non-lysine based dendrimers can also be prepared and areintended to be within the scope of the PEG/PLL dendrimers describedherein. For example, the dendrimers can include polycationic groupsother than amines, for example, quaternary ammonium salts. Further,synthetic, non-amino acid based cations can be included. Cationic aminoacids such as ornithine can also be incorporated into the dendrimers.

[0043] 2. Additional Polymeric Components

[0044] Additional domains, linking groups, and bioactive materials canbe added to this basic two-domain structure. Examples of suitabledomains include bioadhesive molecules, domains which convert from abinding domain to a non-binding domain in vivo, and domains whichconvert from a non-binding domain to a binding domain in vivo. Examplesof suitable linking groups include biodegradable linkages, such asanhydride, ester, amide and carbonate linkages. Examples of suitablebioactive materials include proteins, polysaccharides, organic compoundswith drug activity, and nucleic acids. The domains and/or linkages canconfer adhesion to particular types of cells or molecules or degradationby enzymatic or non-enzymatic means. The domains may be a third type ofpolymer, for example, a biodegradable polymer such as a polyanhydride,polyhydroxy acid or polycarbonate. When serving to direct attachment, apeptide such as RGD, or even a single amino acid, which is used totarget a polyamino acid for cleavage by an enzyme, can be incorporatedinto the polymer structure.

[0045] Photopolymerizable substituents, including acrylates,diacrylates, oligoacrylates, dimethacrylates, or oligomethacrylates, andother biologically acceptable photopolymerizable groups, can also beadded to the polymeric materials. These can be used to furtherpolymerize the polymer once it is in contact with tissue or othersurfaces, which can result in improved adherence to the surface.

[0046] B. Polycationic—Polyanionic Polymer Complexes

[0047] 1. Polycationic Polymers

[0048] The polycationic material can be any biocompatible water-solublepolycationic polymer, for example, any polymer having protonatedheterocycles attached as pendant groups. As used herein, “water soluble”means that the entire polymer must be soluble in aqueous solutions, suchas buffered saline or buffered saline with small amounts of addedorganic solvents as co-solvents, at a temperature between 20 and 37° C.In some embodiments, the material will not be sufficiently soluble(defined herein as soluble to the extent of at least one gram per liter)in aqueous solutions per se but can be brought into solution by graftingthe polycationic polymer with water-soluble polynonionic materials suchas polyethylene glycol.

[0049] Representative polycationic materials include natural andunnatural polyamino acids having net positive charge at neutral pH,positively charged polysaccharides, and positively charged syntheticpolymers. Examples of suitable polycationic materials include polyamineshaving amine groups on either the polymer backbone or the polymersidechains, such as poly-L-lysine and other positively charged polyaminoacids of natural or synthetic amino acids or mixtures of amino acids,including poly(D-lysine), poly(ornithine), poly(arginine), andpoly(histidine), and nonpeptide polyamines such as poly(aminostyrene),poly(aminoacrylate), poly (N-methyl aminoacrylate), poly(N-ethylaminoacrylate), poly(N,N-dimethyl aminoacrylate),poly(N,N-diethylaminoacrylate), poly(aminomethacrylate), poly(N-methylamino-methacrylate), poly(N-ethyl aminomethacrylate), poly(N,N-dimethylaminomethacrylate), poly(N,N-diethyl aminomethacrylate),poly(ethyleneimine), polymers of quaternary amines, such aspoly(N,N,N-trimethylaminoacrylate chloride),poly(methyacrylamidopropyltrimethyl ammonium chloride), and natural orsynthetic polysaccharides such as chitosan. Polylysine is a preferredmaterial. In some embodiments, the polycationic material is covalentlygrafted to a non tissue-binding polymer, and this material is used toform at least one of the multilayers, preferably the topmost orbottommost layer.

[0050] In general, the polymers must include at least five charges, andthe molecular weight of the polycationic material must be sufficient toyield the desired degree of binding to a tissue or other surface, havinga molecular weight of at least 1000 g/mole.

[0051] 2. Polyanionic Polymers

[0052] The polyanionic material can be any biocompatible water-solublepolyanionic polymer, for example, any polymer having carboxylic acidgroups attached as pendant groups. Suitable materials include alginate,carrageenan, furcellaran, pectin, xanthan, hyaluronic acid, heparin,heparan sulfate, chondroitin sulfate, dermatan sulfate, dextran sulfate,poly(meth)acrylic acid, oxidized cellulose, carboxymethyl cellulose andcrosmarmelose, synthetic polymers and copolymers containing pendantcarboxyl groups, such as those containing maleic acid or fumaric acid inthe backbone. Polyaminoacids of predominantly negative charge are alsosuitable. Examples of these materials include polyaspartic acid,polyglutamic acid, and copolymers thereof with other natural andunnatural amino acids. Polyphenolic materials such as tannins andlignins can be used if they are sufficiently biocompatible. Preferredmaterials include alginate, pectin, carboxymethyl cellulose, heparin andhyaluronic acid.

[0053] In general, the molecular weight of the polyanionic material mustbe sufficient to yield strong adhesion to the polycationic material. Thelengths of the polycationic and polyanionic materials which would resultin good blockage of adhesive interactions may be determined by routineexperimentation. It should be understood that “good” is a word that mustbe defined by the requirements of the particular circumstance at hand,e.g., how long binding is required and how complete a repulsion isrequired by the particular medical application.

[0054] 3. Attachment of Bioactive Species

[0055] Bioactive species can be attached to the ends of the polymers,either covalently or ionically, or by mixing the bioactive species withthe polymeric material, preferably before it is applied to the cells ortissue.

[0056] A wide variety of biologically active materials can beencapsulated or incorporated, including proteins such as antibodies,receptor ligands and enzymes, peptides such as adhesion peptides,sugars, oligosaccharides, and polysaccharides, organic or inorganicdrugs, nucleic acids, and cells, tissues, sub-cellular organelles orother sub-cellular components.

[0057] Bioactive species may be used to target adhesion of the polymericmaterial, to effect a biological activity at the polymericmaterial-tissue interface, or to effect an activity when the bioactivespecies is released during degradation of the polymeric material.

[0058] An example of a suitable ligand is the pentapeptideTyr-Ile-Gly-Ser-Arg (YIGSR), which supports endothelial, smooth musclecell, and fibroblast adhesion, but not platelet adhesion; or thetetrapeptide Arg-Glu-Asp-Val (REDV), which has been shown to supportendothelial cell adhesion but not that of smooth muscle cells,fibroblasts, or platelets, as described in Hubbell, et al.,BioTechnology 9:568-572 (1991). YIGSR, from laminin, binds to receptorson endothelial cells, but not on blood platelets. Thus, the conjugationof the oligopeptide YIGSR to the termini of the (A)x and adsorbing thepolymeric material to a damaged vessel wall would be expected to blockthrombosis on the vessel wall but not to block re-endothelializationfrom the surrounding undamaged vessel wall. This embodiment makes itpossible to cover an injured vessel wall to prevent thrombosis but, viaan adhesion ligand on the termini of one or more of the polymericcomponents, to permit the regrowth of endothelial cells upon thepolymer. This approach also permits the re-endothelialization of thevessel wall while it is still not adhesive to platelets, thus enablinghealing while avoiding platelet activation and thrombus formation.

[0059] 4. Formation of Polymeric Multilayers

[0060] Polymeric multilayers can be formed by alternating application ofpolyanions and polycations to surfaces to form coacervated coatings.Multilayers of coacervated polyions can be formed on macroscopic tissuesurfaces, including mammalian tissue surfaces, and thereby providevarious benefits to the coated surfaces. These include the prevention ofadherence of tissue to tissue, or of cells to tissue, or provision ofselective adherence, as described below. Use of macroscopic tissues asthe substrate avoids the problems associated with the coating ofindividual cells or groups of cells as has been done by others, sincethe tissue is generally vascularized and is therefore provided withnutrients, oxygen and waste product removal. The layers can be used toencapsulate, plug, seal, or support a macroscopic surface. Theapplication of a multilayer coating can be used to minimize or preventtissue adhesion, minimize or prevent postoperative adhesions, preventthrombosis, prevent implantation of cancerous cells, coat tissue toencourage healing or prevent infection, or enhance the local delivery ofbioactive agents. Preferably, at least four layers, and, morepreferably, at least six layers are used to form the coatings.

[0061] 5. Assembly of Complexes with Anion-Cation Coupling

[0062] Microscopic structures can be produced by successive incubationof a surface with solutions of polyionic compounds. In this embodiment,a solution including a polycation is applied to a surface, the excesssolution washed off of the surface, and a solution including a polyanionis then applied to the surface. This process is repeated until thedesired thickness is obtained. This process is referred to herein as“multilayer techniques”. If only a monolayer of each polyelectrolyteadsorbs with each incubation, then electrostatically crosslinkedhydrogel-type materials can be built on a surface a few microns at atime. In another embodiment, the surface is not thoroughly rinsedbetween the application of the polycation and the polyanion. This leadsto the formation of thicker, hydrogel-like structures. The multilayerstend to be relatively bioinert without further treatment, and areeffective at preventing cell spreading on an extracellular matrixsurface. The multilayers can be used, for example, to form protectivebarriers during surgery. An apparatus equipped with a spray nozzle canbe used, for example, to spray a layer at a time of a polycationfollowed by a layer of a polyanion. Alternatively, both polyelectrolytescan be sprayed simultaneously to create relatively thicker layers.

[0063] 6. Thickness and Conformation of Polymer Layer.

[0064] Membrane thickness affects a variety of parameters, includingperm-selectivity, rigidity, and size of the membrane. Thickness can bevaried by selection of the reaction components and/or the reactionconditions. When alternating layers of polycationic and polyanionicmaterials are used, the layer thickness can be controlled by adjustingthe number of layers and also the degree of rinsing between layers. Inspraying layers, control of drop size and density can provide coatingsof the desired thickness without necessarily requiring rinsing betweenlayers. Additionally, the excess (unbound) material can be removed viaother means, for example, by an air jet.

[0065] If the residual polyelectrolyte from the previous layer issubstantially removed before adding the subsequent layer, the thicknessper layer decreases. Accordingly, in a preferred embodiment, the surfaceis first coated with a polycation, the excess polycation is removed byrinsing the surface, the polyanion is added, the excess is removed, andthe process is repeated as necessary.

[0066] By increasing the number of cycles, for example, to 50 or higher,the polymer systems can be used to generate thick, non-adhesive films.

[0067] II. Synthesis of Polymeric Materials

[0068] A. Synthesis of PEG/PLL Copolymers

[0069] PEG may be bonded to the famines of lysine residues ofpoly(L-lysine) as follows. Poly(L-lysine) (PLL) can be reacted with aPEG with one end protected (i.e., a protected monomethoxy PEG), theterminal hydroxyl of which has been previously activated withcarbonyldiimidazole (CDI). The PLL and the activated PEG can be mixed inan aqueous solution buffered at pH 9 and allowed to react for 48 hoursat room temperature. The number of PEG chains grafted per PLL chain maybe controlled by adjusting the ratio of moles of activated PEG added permole of added PLL. The reaction may not proceed to completion, i.e., themole ratio of PEG to PLL in the reaction mixture may not be identical tothat in the PEG-b-PLL product, but higher ratios of PEG to PLL willproduce higher amounts of PEG in the PEG-b-PLL product.

[0070] The cationic domains tend to be highly reactive, and efforts mustbe made to control the extent of addition of PEG to PLL. Executing thereaction in the absence of water reduces deactivation of PEG and allowsbetter stoichiometric control. For example, unprotected poly-L-lysinecan be dissolved in water, then added to dimethylformamide (DMF) to makea solution that is 5% aqueous. The poly-L-lysine can then be reactedwith CDI mono-activated PEG in stoichiometric amounts, followed byevaporation of solvent under vacuum yielding a PEG/PLL copolymer.Alternatively, unprotected poly-L-lysine can be dissolved in water andprecipitated by adding NaOH. The precipitated polymer can then be addedto anhydrous DMF and then reacted with CDI mono-activated PEG instoichiometric amounts, yielding an (A)x-b-(B)y copolymer. When thereaction is performed in the absence of water, side reactions involvingthe activated group can be reduced (i.e., deactivation is reduced), andat long reaction times the ratio of mole PLL to PEG in the polymerproduct more closely resembles than in the reactant mixture.

[0071] Solution polymerization of PLL may be carried out using monomerscontaining different epsilon protecting groups, which allows strictcontrol over the degree of substitution of PEG onto PLL. N-carboxyanhydrides of various amino acids may be synthesized and polymerizedinto copolymers, as in the following example.N,N′-dicarbobenzoxy-L-lysine (Z,Z-lysine) can be reacted with phosphoruspentachloride to yield α,N-carbobenzoxy-α,N-carboxy-L-lysine anhydride.α,N-carbobenzoxy-α,N-tert-butyloxycarbonyl-L-lysine (Z,boc-lysine) canbe reacted with sodium methoxide to yield the sodium salt ofZ,boc-lysine. The sodium salt of Z,boc-lysine can be reacted withphosphorus pentachloride to yieldα,N-tert-butyloxycarbonyl-α,N-carboxy-L-lysine anhydride. Z,Z-lysineanhydride can be added to Z,boc-lysine anhydride, and the two monomerscan be polymerized by the addition of sodium methoxide as an initiator.A copolymer results, poly(α boc-lysine)-co-(α Z-lysine). The boc groupscan be removed by addition of the polymer to trifluoroacetic acid forfifteen minutes.

[0072] The salt form can be converted to the free base by reaction witha reactant such as pyridine. The free amines on the polymer can then bereacted with CDI PEG in DMF. The Z groups can then be deprotected byadding the polymer to HBr in acetic acid for fifteen minutes, yieldingan (PEG)x-b-(PLL)y copolymer, where the ratio of PEG to PLL in the finalproduct can be controlled by the initial ratio of boc protected lysines.

[0073] It may be desirable to produce versions of the polymer which arenot of a brush structure. This may be facilitated by not deprotectingthe epsilon amines of PLL, so that the only reactive groups are theamine and carboxyl termini. For example, reaction of CDI mon-activatedPEG with poly α,N-carbobenzoxy-L-lysine in DMF yields an (A)x-(B)ycopolymer. Activation of the carboxyl terminus of the (A)x-(B)ycopolymer with TSU followed by reaction with mono-amino PEG in DMFyields an (A)x-(B)y-(A)z copolymer.

[0074] In some embodiments, it may be desirable to incorporatebiodegradable polymers such as polylactides, polyanhydrides, orpolycarbonates. For example, monomethoxy PEG reacts with d,l-lactide(1:3 molar ratio) in xylene in the presence of stannous octate underreflux for sixteen hours to yield a PEG with an end group which degradesover time in water. The hydroxyl at the terminus of the trilactide endgroup can be activated with CDI, which can then further reacted with PLLby methods presented above to yield an (A)xC-b-(B)y, an (A)xC-(B)y or an(A)xC-(B)y-C(A)z copolymer. Similar nucleophilic displacement chemistrycan be used to couple other biodegradable polymers to the PEG backbone.

[0075] In some embodiments, it may be desirable to incorporate apolymeric material with a non-binding backbone which is converted to abinding backbone through degradable linkages. For example, the terminalamine on polyglutamic acid can be reacted with CDI PEG to produce an(A)x(D)y-b-(B)y copolymer. The copolymer can be dissolved in water at pH2 and lyophilized to convert the carboxylic acid salt to the free acid.The polymer can be dissolved in DMF, and the glutamic acid residuesactivated with TSU. The activated polymer can then be reacted with bocprotected aminoethanol in DMF overnight at room temperature and thendeprotected and desalted. The resulting product is initiallypolycationic and binding, but hydrolyses to a non-binding polyanion.

[0076] In other embodiments it may be desirable to incorporate amaterial which converts over time from a material which is repulsive tocells to one which is non-repulsive to cells. For example, a polypeptidemay be reacted with an (unprotected) hydroxy acid using peptidesynthesis techniques to yield a nonionic polymer. Over time, as theamide linkage degrades, the nonion converts from repulsive to notrepulsive.

[0077] B. Optimization of the Polymeric Material for IndividualApplications.

[0078] The biological performance of these materials can be optimized byaltering the structure of the polymers, the ratio of the number oftissue-binding polymers to non-binding polymers, and the ratio of themass of the tissue-binding polymers to non-binding polymers.

[0079] In some cases, polymeric materials exhibiting more than onemanner of degradation may be required to achieve different results. Forexample, degradation by nonenzymatic hydrolysis will depend primarilyupon the accessibility of the polymeric material to water and the localpH. Given that pH and water concentration are similar throughout manyparts of the body, such a mode of degradation would yield a loss inrepulsiveness of the polymer that depends mostly upon time. As anotherexample, if the degradable region is sensitive to an enzyme, theactivity of which is not highly regulated but rather was present in thebody fluids at a more or less constant level, the rate of loss ofrepulsiveness depends primarily upon time. As another example, if thedegradable region is sensitive to an enzyme, the activity of which ismore highly regulated, the rate of loss of repulsiveness will dependmore upon the expression of that particular enzyme activity. Forexample, many types of cells express the proteases plasmin orcollagenase during migration. Sensitivity to plasmin by the polymerallows the polymer to be degraded by cells migrating onto the surface,so that they can attach to recolonize the surface. This is particularlyuseful in prevention or treatment of restenosis.

[0080] The biological performance of these polymeric materials dependsupon their structure. Specific features of biological performanceinclude binding to the tissue, repulsion of opposing tissues, durationof binding to the tissue, duration of repulsion of opposing tissues, andthe mode of loss of binding or repulsion. Specific features of polymericmaterial structure include the type (chemical composition) oftissue-binding domain, type of non-binding domain, the ratio of the massof binding to non-binding domains, the number of binding to non-bindingdomains, the inclusion of sites that are particularly susceptible tononenzymatic hydrolysis, the inclusion of sites that are particularlysusceptible to enzymatic hydrolysis, and the inclusions of sites withparticular biological affinity.

[0081] C. Method for Forming Polymeric Materials.

[0082] Polymeric objects can be formed into a desired shape by standardtechniques known to those skilled in the art, for example, usingcasting, molding, or solid free form techniques such as threedimensional printing techniques. For example, a mask can be used tocover a specific area in a layer in which a polyelectrolyte is not to beadded. The shape of the object can be controlled as subsequent layersare added.

[0083] The materials may also be shaped in relative to an internal orexternal supporting structure. Internal supporting structures includescreening networks of stable or degradable polymers or nontoxic metals.External structures include, for example, casting the gel within acylinder so that the internal surface of the cylinder is lined with thegel containing the biological materials.

[0084] III. Methods of Use

[0085] The materials have a variety of applications. These include localapplication, either at the time of surgery or via injection into tissue,to prevent adhesion of tissues; to deliver bioactive compounds whererelease is effected more efficiently or at a more desirable rate orwhere tissue encapsulation could detrimentally affect or delay release;to prevent thrombus formation at blood vessel surfaces, for example,following angioplasty; to alter cellular attachment, especially toprevent cellular attachment, and therefore decrease metastasis of tumorcells; and to coat prosthetic implants such as heart valves and vasculargrafts derived from processed tissues.

[0086] As defined herein, “tissue” includes tissues removed from thebody and tissues present in the body, but specifically excludes cellsand cell aggregates, because these may be adversely affected by coatingthem with the polymeric materials. This term can also be applied totreated tissue, such as tissue heart valves, blood vessels andmembranes, where the tissue is no longer living and has been chemicallyfixed, or a cryopreserved blood vessel or other tissue.

[0087] The polymeric materials can be applied directly by localized ortopical application, or can be delivered systemically to deliver drugs.Topical or localized application can be achieved generally by sprayingor injecting a very thin layer (usually on the order of monolayers ofpolymeric material) onto the surface to be coated. Methods for applyingthe polymeric materials in this manner are known to those skilled in theart.

[0088] A. Coating of Non-Biological Surfaces

[0089] The polymeric materials can also be applied to a non-biological,preferably anionic, surface intended to be placed in contact with abiological environment. Such surfaces include, for example, catheters,prosthetics, implants, vascular grafts, contact lenses, intraocularlenses, ultrafiltration membranes, and containers for biologicalmaterials. Additionally, cell culture dishes, or portions thereof, canbe treated to minimize adhesion of cells to the dish. Cell culturedishes treated in this manner only allow cell spreading in those areaswhich are not treated, when the cells are anchorage dependent cells(cells which must be anchored to a solid support in order to spread).

[0090] The polymeric materials can be applied to the treatment ofmacrocapsular surfaces, such as those used for ultrafiltration,hemodialysis and non-microencapsulated immunoisolation of animal tissue.The surface may be in the form of a hollow fiber, a spiral module, aflat sheet or other configuration.

[0091] B. Coating of Metal and Ceramic Surfaces

[0092] Metal surfaces in contact with biological fluids can be coatedwith the polymeric materials. Absent such a coating, these surfaces canbe quickly fouled by adsorption of a protein layer when in contact withbiological fluids. Deposition of biological matter is minimized bycoating the surfaces with the polymeric materials.

[0093] Examples of protein-repelling water soluble polymers include, butare not limited to, polyethylene glycol, polyethylene oxide,poly-N-vinyl pyrrolidone, polyhydroxyethyl methacrylate, and polyacrylicacid. The polycationic polymers include, but are not limited to,polylysine, polyarginine, and polyethylenimine.

[0094] The metals which are passivated are those that present a netanionic metal or metal oxide surface in water at physiological pH, andare used to form conduits for the flow of biological fluids, or thoseused to form devices in contact with biological fluids, including thosedevices. that are implanted into humans or animals. The metals includestainless steel and titanium, or surfaces with metal oxides such as ironoxide, titanium oxide and silicon oxide. The metals are treated with thepolymer as a part of conduit or device manufacture, or are treated insitu, following assembly of the conduit or device, or as part of thenormal operation of the device.

[0095] The treatment may be. applied by adsorption of the polymericmaterials from a liquid solution, or by spraying. Removal of the polymerfrom the metal via a change in pH, or other means, may also be. used asa cleaning step as a part of the normal operation of a device using thistechnology, as well as the reapplication of a polymer layer to themetal, resulting in a resumption of the protein-repelling properties atthe treated metal surface.

[0096] The compositions and methods described herein will be more fullyunderstood with reference to the following non-limiting examples.

EXAMPLE 1 Effect of Materials on Cell Spreading

[0097] A variety of methods can be used to optimize the desiredproperties. For example, when a PEG-b-PLL brush copolymer is used toprotect an injured tissue surface from the adhesion of cells approachingfrom the fluid phase in contact with that tissue surface, the polymericmaterial can be optimized by conducting studies using a tissue culturemodel.

[0098] Fibroblasts can be seeded on a multiwell dish treated withproteins adsorbed from cell culture medium containing 10% serum. Some ofthe wells can be coated with the copolymers, and others can be leftuncoated. Then, the surface can be seeded with fibroblasts in culturemedium containing 10% serum and the adhesion and spreading can bemonitored.

[0099] A measurement of the fraction of cells adhering (Fa) and fractionof cells spreading (Fs) may be made based on morphological criteriausing light microscopy. Such measurements conducted 4 hours followingseeding can provide useful measures of adhesion and repulsion.

[0100] The effect of cell spreading is localized to a surface. Forexample, when PEG/PLL is spread on a portion of a surface, only thatportion of the surface resists cell adhesion. A PEG/PLL solution wasspread on approximately one half of a polystyrene cell culture well.After seeding the well with fibroblast cells, cells spread on theuntreated area of the well, but did not spread onto the treated half ofthe well. A sharp transition was observed between the untreated andtreated regions.

[0101] When human foreskin fibroblast cells (HFF cells) were added to atissue culture well coated with PEG/PLL, they did not spread. However,when the cells were transferred to an untreated well, cell spreadingproceeded normally on the untreated surface.

[0102] Red blood cells can be aggregated with wheat germ agglutinin. Theability of various types of PEG/PLL polymers to minimize agglutinationwas evaluated. PEG/PLL copolymers with a 5 Kd PEG chain and a 375 Kd PLLchain, with graft ratios of 14, 10.5, 7.5, 7, 5.6, 3.5, 1.75 and 1.25were prepared and evaluated. The PEG/PLL polymers with a graft ratio of14 agglutinates RBC (even without the addition of wheat germagglutinin), 10.5 agglutinates some of the red blood cells, and protectssome of the red blood cells from wheat germ-agglutinin inducedhemagglutination, 7.5 forms a complex, 7 hinders agglutination, 5.6hinders agglutination, 3.5 slightly hinders agglutination, and 1.75 and1.25 have no effect on agglutination. Accordingly, a useful range ofgraft ratios for these polymers for minimizing agglutination of RBC isbetween 3.5 and 10.5.

[0103] In contrast, PEG/PLL with a 5K PEG chain and a 20 KD PLL chainhad either no effect on agglutination (graft ratios of between 1.75 and7) or caused agglutination (graft ratios of 10.5 and 14). The resultsare summarized in Table 1. TABLE 1 Effect of PEG/PLL on Agglutination ofRed Blood Cells Highest Highest conc. of conc. of Highest conc. WGA withWGA of WGA pellet without large without small Polymer formationaggregates aggregates Comments PBS 1.54 0.514 0.514 No effect mPEG 5K1.54 0.514 0.514 No effect PLL 20K Agglut Agglut Agglut Agglut RBC PLL418K Agglut Agglut Agglut Agglut RBC Plu F-68 1.54 0.514 0.514 No effect5/20 - 1.75 1.54 0.514 0.514 No effect 5/20 - 3.5 1.54 0.514 0.514 Noeffect 5/20 - 7 1.54 0.514 0.514 No effect 5/20 - 10.5 0.514 0.171Agglut Agglut RBC 5/20 - 14 Agglut Agglut Agglut Agglut RBC 5/375 - 1.251.54 0.514 0.514 No effect 5/375 - 1.75 1.54 0.514 0.514 No effect5/375 - 3.5 1.54 0.514 0.514 No effect 5/375 - 5.6 125 125 125 Hindersagglut. by WGA, slightly 5/375 - 7 125 125 13.9 Hinders agglut. 5/375 -7.5 125 125 Agglut Complex 5/375 - 10 125 125 125 Hinders agglut.5/375 - 10.5 125 1.54 1.54 Complex 5/375 - 14 Agglut Agglut Agglut.Agglut RBC

[0104] In Table 1, PBS indicates phosphate buffered saline. mPEG 5Kindicates monomethoxy PEG with a molecular weight of 5K. PLL 20Kindicates a polylysine with a molecular weight of 20K, whereas PLL 418Kspecifies that the molecular weight of the PLL is 418 K. Plu F-68 is aspecific pluronic nonionic surfactant. 5/20 indicates that the polymerincludes a PEG with a molecular weight of SK, and PLL with a molecularweight of 20K. 5/375 indicates that the polymer includes a PEG with amolecular weight of 5K, and PLL with a molecular weight of 375K. Thenumbers following 5/20 or 5/375 specify the ratio of grafting of PEG toPLL.

[0105] An adsorbed serum protein substrate was incubated with PEG/PLL5/20 and PEG/PLL 5/375. The polymers with relatively low molecularweight PLL did not lead to any change in the response of the cells tothe surface. However, the series of copolymers with the higher molecularweight PLL led to a significant reduction in cell spreading on theadsorbed serum protein substrate as PEG molecules were added to the PLLbackbone. Cell spreading was eliminated when the graft ratio was 7 or3.5 and was significantly lower relative to the low molecular weight PLLwhen the graft ratio was 10.5. The results are shown in FIG. 3.

[0106] The data show that PLL-g-PEG polymers with a relatively lowmolecular weight PLL backbone allow cell spreading and those with arelatively high molecular weight PLL backbone prevent cell spreading.

EXAMPLE 2 Evaluation of PEG/PLL Dendrimers

[0107] Various model surfaces were treated with the PEG/PLL, where thePLL is in the form of dendrimers, then washed with PBS. Fibroblasts werethen seeded in serum containing media, and the number of spread cellsper square millimeter was counted at four hours. The results are shownin Tables 2 (a-c). The results show that as the number of amine groupsis increased to 4 or more, the number of spread cells is significantlydecreased. TABLE 2 Cell Spreading on TCPS Coated with PEG/PLL dendrimersDendron Generation (# of Amines) PEG 20K PEO 100K PEG 5K —OH (0) 144 ±13  83 ± 33 150 ± 18  Y (1) 125 ± 22  147 ± 21  118 ± 37  1 (2) 137 ±33  92 ± 39 132 +/− 8  2 (4) 0 ± 0 33 ± 7  — 3 (8) 0 ± 0 0 ± 0 1 ± 1 4(16) 0 ± 0 3 ± 3 — 5 (32) 0 ± 0 — —

[0108] In Table 2, -OH indicates that the polymer is PEG, with 20K,100K, and 5K representing the molecular weight of the polymers. Yindicates a zero generation dendrimer, in which one amino group ispresent.

[0109] The results demonstrate that, at least with respect to TCPSsurfaces, dendrimers with between 4 and 32 amine groups significantlyreduced cell spreading. In all cases, spreading was reduced as thegeneration of the PLL dendrimer increased.

[0110] The polymers were used to assess the immobilization of PEG to thebiological surfaces through adsorption of the polycationic block. ThePEG dendrons were able to prevent cell spreading on a simple anionicsurface, indicating that PEG was present on the surface, however, thecopolymers had no effect on cell spreading on proteinaceous surfaces.Treatment of red blood cells with PEG 20K with a five generation lysinedendron (32 amines) was able to hinder hemagglutination by a lectin,however, the polymer itself was found to agglutinate red blood cells inthe absence of mixing.

EXAMPLE 3 Cell Spreading on Polyelectrolyte Multilayers

[0111] HFF Cells On TCPS

[0112] In order to establish a baseline for cell spreading onpolyelectrolyte multilayers, multilayers were formed onto tissue culturepolystyrene (TCPS) using 0.1% PLL (MW 50,000) and 0.15% alginate, with 4PBS washes following each adsorption step, out to 15 bilayers. On thissurface, the number of well spread cells dropped to zero after thesecond layer was added (see FIG. 1A). However, quite a few poorly spreadcells were observed on the substrates with 2 to 15 layers, with theirnumber remaining somewhat constant out to 15 layers.

[0113] HFF Cells on Gelatin

[0114] Gelatin is produced by denaturing collagen, and substrates fortissue culture are often coated with gelatin to enhance cell attachmentto a substrate. As a denatured protein, a mixture of hydrophobic andhydrophilic residues are exposed, and a very heterogeneous surfaceshould be generated upon adsorption to a surface.

[0115] Using 0.1% PLL and 0.15% alginate, multilayer structures wereformed on the gelatin coated tissue culture polystyrene substrates, with4 deionized water washes between adsorption steps. HFF adhesiondecreased on substrates with more layers, and was inhibited afterformation of ten layers on the substrate (see FIG. 1B).

[0116] When a polyethylene imine and polyacrylic acid (PEI and PAA) (MW50,000 for both polymers) were used in place of PLL and alginate, thenumber of spread cells decreased as the number of layers increased.However, the extent of lowering of cell spreading was less than thatobserved when PLL and alginate were used. The results are shown in FIG.1D.

[0117] HFF Cells on HFF ECM

[0118] An extracellular matrix of fibroblasts on a surface is a goodmodel for the surface of a damaged tissue. Fibroblasts depositfibronectin and collagen, and many other proteins, while growing on asurface, making very cell adhesive surfaces, after the cells are removedwith 0.1 N ammonium hydroxide. The presence of fibrillar collagen on thesurface would greatly increase the surface roughness of the adsorbedlayer, since the network of fibrils are large enough to be seen at100×by phase contrast microscopy.

[0119] A surface was coated with human foreskin fibroblast extracellularmatrix (HFF ECM). A coating was prepared by sequentially addingalternating layers of PLL and alginate, using 0.1% PLL and 0.15%alginate solutions, and washing the surface with 4 deionized waterwashes between adsorption steps. The spreading of HFF cells was greatlyinhibited after 5 layers were applied to the surface, however, poorlyspread cells were found even after 15 layers were applied. These resultsare similar to the results obtained with TCPS (see FIG. 1C).

[0120] Toxicity

[0121] The inhibition of cell spreading at hydrogel surfaces can beexplained in terms of limited interaction of proteins with the hydrogelsurface. Ideally, no other metabolic activities of the cells should beaffected by the surface, and thus the cells in contact with the surfaceshould remain viable and competent for cell spreading for up to 24 h.Polycations are known to be toxic to cells at low concentrations, andthus the effects could be a result of cellular toxicity. Therefore,viability and cell spreading were assessed for HFF cells on the TCPSsubstrate with 15 PLL/alginate bilayers after one hour. By addingfluorescein diacetate to the cell culture media, metabolically activecells become labeled with fluorescein by the function of esterases inthe cell. The fluorescently labeled cells were counted, and it was foundthat 96.3% were fluorescent, indicating viability and metabolic activityafter 1 hr on the multilayer surface. At this time point, cell spreadinghad commenced on the untreated surfaces and on surfaces with only a fewbilayers, but no cells were spread on the surface with 15 bilayers.

[0122] After one hour in a TCPS well with 15 PLL/alginate bilayers, thecells in the well were moved to a new well which was untreated TCPS. Thecells were moved by pipeting the media from the first well into thesecond well, without the addition of new media. Normal cell spreadingwas observed, and only well spread cells were found at 24 hours.

[0123] Additionally, a multilayer assembly formed using 1% PLL and 1.5%alginate, with 15 min. incubation times and with only one PBS wash, wasformed in a tilted tissue culture well, such that only half of the wellwas treated. Two bilayers were formed, and cells seeded onto the well.Cells spread normally on the untreated side, and spread and migratedright up to edge of the multilayer. Cells on the side with themultilayer did not spread. The multilayers formed using these conditionsare very thick, and thus toxicity due to the leaching of polymer wouldbe more prominent in this system. The spreading of cells on theuntreated side indicated that cell spreading is not inhibited in thepresence of polyelectrolyte multilayers by the release of a soluble,toxic factor.

[0124] The presence of phenol red in tissue culture media allows the pHto be monitored during an experiment. No change was detected in thecolor of the tissue culture medium as compared to control substrates.

EXAMPLE 4 Coating of Si/SiO₂ Wafers with Polyelectrolytes

[0125] Solutions containing 0.1% PLL (MW?) and 0.15% alginate, both inPBS at pH 7 were used in multilayer techniques to produce self-assembledstructures on Si/SiO₂ wafers and Si/SiO₂ wafers with adsorbed gelatin.As the number of bilayers increased, so did the thickness of the layer.The thickness of the bilayers (as measured by ellipsometry) as a resultof the number of bilayers is shown in FIG. 2. The ellipsometric datawith the PLL and alginate system suggests that the thickness of thepolymeric layers does not increase linearly.

[0126] As further evidence for formation of multilayer structures on agelatin coated substrate, dynamic contact angles were measured asmultilayers were formed on a clean glass surface coated with gelatin, asa method for detecting changes in surface chemistry. The advancing andreceding contact angles for the clean glass surface were 13.4 and 11degrees, respectively. After adsorption of gelatin, the advancing andreceding contact angles were 46.4±7.0 degrees and 17.3±4 degrees,respectively. Using 0.1% PLL, and 0.15% alginate in PBS at pH 7, withfour deionized water washes following the adsorption of eachpolyelectrolyte layer, the receding contact angle gradually decreased tounder 10 degrees, but the advancing contact angle changed from about 60degrees following deposition of a polylysine layer to about 40 degreesfollowing deposition of an alginate layer. In the dry state, polylysinewould be expected to be somewhat hydrophobic, since polylysine'shydrophilicity comes mainly from its charged amines, which would beneutral or exist as a salt in the dry state. Alginate, having numeroushydroxyl groups, would be expected to be more hydrophilic in the drystate. This data indicated that the surface of a gelatin coatedsubstrate was being changed with each polyelectrolyte adsorption step.After addition of four bilayers of polymer, the samples became highlywettable, such that water would not readily retract from the surfacefollowing removal from water. One sample was built up to 15 bilayers. Atthis point, for the polylysine layer, the contact angles were 80 degreesand 7.6 degrees, advancing and receding, respectively, and for thealginate layer, the contact angles were 64.2 and 6.98 degrees.

[0127] The data indicated pairing in the progression of advancingcontact angle of each gelatin substrate, and thus the change inadvancing contact angle from one step to the next for each substrate wasanalyzed. Statistical significance was judged for each adsorption stepin comparison with the previous adsorption step using ANOVA techniques.After adding the third alginate layer, the change in contact angle fromone layer to the next was significant at a confidence level greater than99.9%.

[0128] In order to correlate the cell spreading behavior with multilayerthickness, PEI and PAA multilayers were built onto Si/SiO₂ substrates,and thicknesses were measured by ellipsometry. Very little change inthickness was observed between 1 and 10 layers, however, a thickness of120 Angstroms was observed at 15 layers, with a thickness per layer of6.7 Angstroms. The thickness at 15 layers with the PEI/PAA system wassimilar to that observed with 5 layers in the PLL/alginate system. Inboth cases, the number of well spread cells was decreased to about athird of the spreading found on gelatin substrate alone. Accordingly,there is a correlation between spreading on the surface and thethickness of the polyelectrolyte coating.

[0129] Surface films prepared using the PEG/PLL copolymers and thealternating layers of polycations and polyanions are highly effective atcoating and preventing cell spreading on even the most adhesive ofsurfaces, such as collagen Type 1, gelatin, fibroblast extracellularmatrix, and fibronectin. The choice of polyelectrolytes isimportant—polylysine/alginate exhibits exponential growth thickness, andis effective in these models when the film thickness are greater thanabout 100 nm. In contrast, when the layers were prepared frompolyethylene imine/polyacrylic acid, the thickness of the layer grewlinearly rather than exponentially, resulting in thinner films per cycleof polycation/polyanion addition. The thickness of the layer correlatedto the degree of minimization of cell spreading, so more layers arenecessary to achieve the same results seen with the PLL/alginate layers.

EXAMPLE 5 Thickness of Multilayer Films is Affected by ProcessingConditions

[0130] PLL and alginate multilayers tend to grow in an exponentialrather than linear fashion. Other polymer layers tend to grow in a morelinear fashion. A series of studies Were conducted to explain thebehavior of the PLL/alginate system, and to determine if the phenomenoncould be generalized to other polymers.

[0131] In the formation of PLL and alginate microcapsules, wallthicknesses on the order of microns can be generated with only threepolymer layers. In this case, the alginate is first gelled into amicrosphere using Ca⁺⁺, and the microspheres are placed into a solutionof PLL. After washing, the microsphere is then added to a solutioncontaining alginate, followed again by washing. A calcium chelator thendissolves the alginate interior of the microcapsule, and microcapsuleswith wall thicknesses on the order of microns result. A simple massbalance leads to the conclusion that a significant mass of PLL must belocalized to the microsphere surface during the adsorption step, if itis assumed that the polymers do not self-associate. This amount of PLLcould be localized to the surface of the alginate microsphere if the PLLwere either to diffuse a certain distance into the alginate microsphereduring the adsorption step, or if a PLL rich, viscous surface layerformed, which resisted washing, and which then gelled upon addition ofalginate.

[0132] With PLL and alginate multilayers, the thickness of the formedmultilayer structures was found to be a function of how well the surfacewas washed during multilayer formation, and the concentration ofpolymer. The thickness of polymer on an Si/SiO₂ substrate followingmultilayer formation was measured using ellipsometry. Thorough washingof substrates led to per layer growth on the order of tens of angstroms.However, in the specific case of 1% PLL and 1.5% alginate, less vigorouswashing led to the growth of macroscopic hydrogels, with dry polymerthicknesses on the order of hundreds of angstroms per layer (See Table3). All treated substrates became much more hydrophilic after theadsorption of the second polymer layer, and remained transparent afterdrying.

[0133] Ellipsometry was used to measure the effect of washing conditionson the ultimate thickness of polyelectrolyte multilayer assemblies. Lessvigorous washing using the PLL/alginate polyelectrolytes led to an orderof magnitude increase in the thickness of polymer on the substrate.TABLE 3 Thickness of PLL/Alginate Layers Thickness (A) ± Treatment Std.Dev. Oxide layer 22.9 ± 1.6 1% PLL, 1.5% alg (15 min treatment, 1  801.6± 223.4 min PBS wash) × 2 1% PLL, 1.5% alg (15 min treatment, 43.9 ± 1.430 sec wash in running water) × 2 0.1% PLL, 0.15% alg (5 min treatment,80.6 ± 8.2 1 min PBS wash) × 2 2% PEI, 2% PAA (15 min, 1 min PBS  89.8 ±11.5 wash) × 2 2% PEI, 2% PAA (2 min, 3 × 1 min 59.9 ± 8.8 PBS wash) × 2

[0134] “Thick” multilayer structures can be formed which are clear andtransparent. When 1.4% alginate is placed into a small well, and 1% PLLis layered on top, a clear complex gel forms at the interface, which canbe removed from the well with a spatula. However, layering of 0.1% PLLon top of 1.4% alginate does not yield a gel, only a precipitate.Substitution of PEI for PLL also leads only to the formation of aprecipitate with 1.4% alginate. Layering of 2% PEI onto 2% PAA 50K doesnot lead to complex gel formation, however, layering 2% PEI onto 25% PAA50K at pH 3, leads to the formation of a clear gel. Thus, by the properchoice of viscosities and charge densities, three dimensional gels canbe formed upon mixing of polyelectrolytes, as opposed to the formationof a simple precipitate or flocculate. The results are summarized inTable 4. TABLE 4 Effect of Various Treatments on the Formation ofPLL/alginate layers Treatment Polymers Result Direct mixing 1.4% alg, 1%PLL Precipitate only 1.4% alg, 0.05% Precipitate only PLL 2% PAA, 2% PEIPrecipitate only 25% PAA, 2% PEI Precipitate only Layering a solution of1.4% alg, 1% PLL Clear gel a polycation onto a 1.4% alg, 0.1%Precipitate only solution of a PLL polyanion 1.4% alg, 2% PEIPrecipitate only 1.4% alg, 1% PEI Precipitate only 2% PAA, 2% PEIPrecipitate only 2% PAA, 1% PLL Precipitate only 25% PAA (pH 3), Cleargel, with 2% PEI cloudy edges 25% PAA (pH 7), Cloudy gel 2% PEI 25% PAA,50% Slightly cloudy gel PEI

[0135] HFF ECM, Treated with “Thick Multilayers”

[0136] Conditions which lead to the formation of “thick” multilayers, asjudged by ellipsometry, yield very thin, clear, transparent surfacefilms on substrates, which can be seen microscopically, and which can bepealed away from the substrate with a spatula (demonstrating significanthorizontal mechanical integrity), but folding in on itself as it ispulled away from the surface, forming an opaque mass.

[0137] Using 1% PLL and 1.5% alginate, with 15 min incubation times,with one PBS wash following each adsorption step, multilayers wereformed onto the HFF ECM surface, and cell adhesion to the substratesTABLE 5 Number of Spread Cells on Polyelectrolyte Layers Number ofSpread Treatment Cells/mm² (± S.D.) 1% PLL/1.5% alg 0 layers Monolayer 1layer 0 ± 0 2 layers 0 ± 0 2% PEI/2% PAA 0 layers Monolayer 1 layersMonolayer 2 layers Monolayer

EXAMPLE 6 Cell Spreading on a Silicon Dioxide Surface

[0138] Cell spreading is indicative of protein adsorption on a surface.Various comb copolymers were applied to a silicon oxide surface and cellspreading on the coated surfaces was evaluated. The polymers werePLL-g-PEG comb copolymers, with the ratios of PEG to lysine units asshown below in Table 1. As shown in Table 1, coating the surface withvarious polymeric materials is able to prevent cell spreading, and cantherefore prevent protein adsorption. TABLE 6 Cell Spreading on a SiO₂Surface Number of spread cells Copolymer (cell/mm²) PLL 98 ± 27PLL-g-PEG (PEG MW 1 ± 1 5000, PLL MW 20,000, 1 PEG per 14 lysine units)PLL-g-PEG (PEG MW 0.4 ± 0.7 5000, PLL MW 20,000, 1 PEG per 10.5 lysineunits) PLL-g-PEG (PEG MW 3 ± 5 5000, PLL MW 20,000, 1 PEG per 7 lysineunits) PLL-g-PEG (PEG MW 0 ± 0 5000, PLL MW 20,000, 1 PEG per 3.5 lysineunits) PLL-g-PEG (PEG MW 190 ± 156 5000, PLL MW 20,000, 1 PEG per 1.75lysine units) PEG 157 ± 70 

[0139] As shown in Table 6, PEG/PLL polymers with specific graft ratiosof PEG per lysine subunits were able to prevent cell spreading, whereasother PEG/PLL polymers with different graft ratios were ineffective.

EXAMPLE 7 Cell Spreading on Multilayer Films

[0140] Peptides were attached via their N-terminal amines to thecarboxyl side chains of polyacrylic acid (MW 250,000) (PAA) withN,N,N′,N′-tetramethyluronium tetrafluoroborate (TSTU). Activated PAA wasprepared by combining 200 μl of 20 mg/ml PAA in anhydrous DMF with 40 μlof 50 mg/ml TSTU and 20 μl of di-isopropyl ethylamine (DIPEA) to achievea mixture with 61 μmol PAA COOH moieties, 6.7 μmol TSTU and 121 μmolDIPEA. A solution of peptide in buffer was added dropwise to theactivated PAA. The peptide-PAA crude mixture was purified of unboundpeptides by dialysis. The samples were lyophilized.

[0141] This process was performed as described with the Arg-Gly-Asp(RGD-), Arg-Asp-Gly (RDG-), Tyr-Ile-Gly-Ser-Arg (YIGSR-), an His-Ala-Val(HAV-) containing peptides.

[0142] Polyelectrolyte multilayers were made within the wells of 24-welltissue culture polystyrene dishes by adsorbing alternating layers ofpolyethylene imine (MW 50,000) (PEI) and PAA or peptide-PAA. The firstlayer of PEI was deposited by adding 0.4 ml PEI (20 mg/ml) in pH 10.0HEPES buffer to a well. After 2 minutes, the well was rinsed three timeswith 0.5 ml PBS for two minutes each rinse. The second layer wasdeposited by adding 0.4 ml of PAA (20 mg/ml) in pH 10.0 PBS. The wellwas rinsed and a second layer of PEI was deposited and rinsed asdescribed above. The final layer was deposited by adding 200 μl of PBSplus 0.1 μmol of peptide-PAA (where the peptide-PAA concentrations aredescribed in terms of the amount of peptide) or PAA in 30 μl ofdeionized water. After 30 minutes, 0.4 ml of PAA (20 mg/ml) was addedand the wells were rinsed after 5 minutes.

[0143] Fibroblast cells were seeded onto polyelectrolyte multilayers ata density of 1000 cells/ml². After 14 hours incubation, the multilayerwith the top layer of PAA attached to the RGD peptide led to 12,500±1500spread cells/cm², while the control (non-sense peptide peptide RGDattached to PAA led to 2500±500 spread cells cm². The data demonstratesthat peptides can be placed on the surface of a polyelectrolytemultilayer assembly.

We claim:
 1. Block or graft copolymers comprising a polycationic blockand at least one non-tissue binding blocks wherein the polycationicblock is either a substantially linear polycationic block with amolecular weight of 100,000 Daltons or more, or a denfritic polycationicblock with a molecular weight sufficient to provide at least 8 cationiccharges.
 2. The copolymer of claim 1, wherein the non-tissue-bindingblock has a molecular weight in excess of 5,000 daltons.
 3. Thecopolymer of claim 1, where the non-tissur-binding block has a molecularweight in excess of 50,000 daltons.
 4. The copolymer of claim 1 whereinthe non-tissue-binding block is selected from the group consisting ofpolyethylene glycol, mixed polyalklene oxide having a solubility of atleast one gram/liter in aqueous solutions, neutral water-solublepolysaccharides, polyvinyl alcohol, poly-B-vinyl pyrrolidone,non-cationic poly(meth)acrylates and combinations thereof.
 5. Thecopolymer of claim 5 wherein the non-tissue-binding block comprisespolyethlene glycol.
 6. The copolymer of claim 1 wherein the polycationicblock is selected from the group consisting of natural and unnaturalpolyamino acids having net positive charge at neutral pH, positivelycharged polysaccharides, and positively charged synthetic polymers. 7.The copolymer of claim 1 wherein the polycationic block comprisesmonomeric units selected from the group consistion of lysine, histidine,arginine and ornithine.
 8. The copolymer of claim 7 wherein thepositively charged polysaccharide is selected from the group consistingof chitosan, partially deacetylated chitin, and amine-containingderivatives of neutral polysaccharides.
 9. The copolymer of claim 6wherein the positively charged syntheic polymer is selected from thegroup consisting of polyethyleneimine, polyamino(meth)acrylate,polyaminostyrene, polyaminoethylene, poly(aminoethyl)ethylene,polyaminoethylstyrene, and N-alkyl derivatives thereof.
 10. Thecopolymer of claim 1 further comprising a pharmaceutically acceptablecarrier.
 11. The copolymer of claim 1 further comprising a bioactiveagent.
 12. The copolymer of claim 11 wherein the bioactive agent ischemically coupled to the polymer.
 13. A polymeric coating on amacroscopic surface, comprising layers of polycationic and polyanionicmaterials.
 14. The coating of claim 13, wherein the coating is appliedas alternation polycationic and polyanionic layers.
 15. The coating ofclaim 13, wherein there are at least 4 layers.
 16. The coating of claim13, wherein there are at least 6 layers.
 17. The coating of claim 13,wherin the polycation is selected from the group consisting of naturaland unnatural polyamino acids having net posistive charge at neutral pH,positively charged polysaccharides, and positively charged syntheicpolymers.
 18. The coating of claim 17, wherein the polycation comprisesmonomeric units selected from the group consisting of lysine, histidine,arginine and ornithine.
 19. The coation of claim 13, wherein thepolyanion is selected from the group consisting of alginate,carrageenan, furcellaran, pectin, xanthan, hyaluronic acid, heparin,heparan sulfate, chondroitin sulfate, cellulose, carboxymethyl celluose,crosmarmelose, syntheic polymers and copolymers containing pendantcarboxyl groups, polyaminoacids of predominantly negative charge, andbiocompatible polyphenolic materials.
 20. The coating of claim 19,wherein the polyanion is selected from the group consising of alginate,pectin, carboxymethyl cellulose, heparin and hyaluronic acid.
 21. Thecoating of claim 13 further comprising a bioactive agent.
 22. Thecoating of claim 21 wherein the bioactive agent is chemically coupled toat least of the polymers.
 23. The coating of claim 13 wherein thesurface is a tissue surface.
 24. The coating of claim 13 wherein thesurface is a surface of a medical device.
 25. The coating of claim 13wherein at least one layer of polyanionic or polycationic polymerfurther comprises a covalently bound non-tissue-binding block.
 26. Thecoating of claim 25 wherein the non-tissue-binging block is in at leastthe outermost layer.
 27. A method for encapsulating, plugging, sealing,or supporting a macroscopic surface, comprising depositing successivelayers of polycationic and polyanionic material on the surface.
 28. Themethod of claim 27, wherein the deposition of the layers of polycationicand polyanionic materials minimizes or prevents tissues adhesion,minimizes or prevents postoperative adhesion, prevents thrombosis,prevents implantation of cancerous cells, coats tissue to encouragehealing or prevent infection, or enhances the local delivery ofbioactive agents.
 29. The method of claim 27, wherein the polycationicmaterials are selected from the group consisting of poly(D-lysine),poly(ornithine), poly(arginine), poly(histidine), poly(aminostyrene),poly(aminoacrylate), poly (N-methyl aminoacrylate), poly(N-ethylaminoacrylate), poly(N,N-dimethyl aminoacrylate),poly(N,N-diethylaminoacrylate), poly (N,N-diethyl aminomethacrylate),poly(ethylene imine), polymers including quaternary amine groups, andnatural or synthetic cationic polysaccharides.
 30. The method of claim27, wherein the polycationic materials are selected from the groupconsisting of polylysine, polyornithine and polyethylene imine.
 31. Themethod of claim 27, wherein the polyanionic materials are selected fromthe group consisting of alginate, carrageenan, furcellaran, pectin,xanthan, hyaluronic acid, heparin sulfate, chodroitin sulfate, dematansulfate, dextran sulfate, poly(meth)acrylic acid, oxidized cellulose,carboxymethyl cellulose, crosmarmelose, synthetic polymers and copolyerscontainings pendant carboxyl groups, polyaminoacids of predominantlynegative charge, and biocompatible polyphenolic materials.
 32. Themethod of claim 27, wherein the polyanionic material is alginate. 33.The method of claim 27, wherein the material further comprises abioactive agent.
 34. The method of claim 33, wherein the bioactive agentis chemically coupled to one or more of the polymers.
 35. The method ofclaim 28 wherein the site where adhesion is to be prevented is a regionwhere tissue has been injured.
 36. The method of claim 28 wherein thesite wherein adhesion is to be prevented has been surgically cut. 37.The method of claim 28 wherein the site where throbosis or adhesion isto be prevented is the lining of s blood vessel that has been damaged.38. The method of claim 28 wherein the tissue to be prevented fromattaching are cancerous of tumor cells.
 39. The method of claim 28wherein the tissue is an organ or lumen of the body which contants otherorgans that have also been injured.
 40. The method of claim 28 whereinthe tissue has been processed.
 41. A method for coating a non-biologicalsurface comprising applying to the surface a copolymer or sequentiallayers of polycationic and polyanionic materials, wherein: the copolymerincludes a polycationic block and at least one non-tissue binding block,wherein the polycationic block is either a substantially linearpolycationic block with a molecular weight of 100,000 Daltons or more,or a dendritic polycationic block with a molecular weight sufficient toprovide at least 8 cationic charges.
 42. The method of claim 41, whereinthe surface is a metal surface.
 43. The use of the coating of claim 13for treatment of a medical condition.
 44. The use of the coating ofclaim 13 for coating the surface of a medical device.
 45. The use of thecopolymer of claim 1 for treatment of a medical condition.
 46. The useof claim 45 wherein the medical treatment is selected from the groupconsisting of minimization or prevention of postoperative adhesions,minimization or prevention of thrombosis, a tissue surface to encouragehealing, coating of a tissue surface to prevent infection, and applyinga coating to enhance the local delivery of drugs.
 47. The use of thecopolymer of claim 1 for coating the surface of a medical device.